Possible relationships between tumorigenicity and expression of beta-GalNAcT-2 and alpha-GalNAcT-3 activities involved in Forssman GSL biosynthesis and SAT-3 in NeuAc-nLcOse4Cer biosynthesis will be a major emphasis of this research project. The overall immediate objectives of this research program are to purify two glycolipid N-acetylgalactosaminyltransferases from guinea pig tumor cell lines 104Cl (tumorigenic) and 106B (nontumorigenic) and to characterize the exact chemical structure (linkage) produced in the enzymatic products. HPLC and enzymatic studies will also be carried out to compare the synthesis of Forssman antigen (glycosphingolipid) during the growth cycle of guinea pig 104Cl and 106B cultured cells. Separation and purification of beta-GalNAcT-2 (UDPGalNAc:GbOse3Cer) (beta-1-3)N-acetylgalactosaminyltransferase) and alpha-GalNAcT-3 (UDP-GalNAc: GbOse4Cer (alpha-1-3)N-acetylgalactosaminyltransferase) will be achieved by DEAE-cellulose and substrate-bound affinity gel column chromatography. Radioactive products from [3H]GbOse3Cer and [14C]Ac-GbOse4Cer will be isolated after reaction with unlabeled UDP-GalNAc. The exact chemical linkages of the terminal GalNAc residues will be determined after permethylation of purified enzymatic products and characterization of the partially methylated radioactive GalNAc units obtained by autoradiography. Anomeric linkages will be determined by using highly purified papaya beta-hexosaminidase and clam alpha-hexosaminidase. Kinetic parameters of solubilized betaGalNAcT-2 and alpha-GalNAcT-3 will be determined. Differential inhibition of these two enzyme activities will be studied in the presence of UDP, IDP, p-chloromercuribenzoate and N-ethylmaleimide. Biosynthesis in vitro and structure determination of sialosyl-neolactotetraosylceramide (NeuAc-nLc-Ose4Cer) in mouse N-18 clone and human neuroblastoma IMR-32 cells will be investigated in the second and third year of the project. These glycolipids have recently been implicated as tumor antigens. Binding of 125I-labeled GSLs, lectin (Euonymus europeus) and toxins (cholera toxin and ricin) to neuroblastoma cell surfaces will also be studied before and after chemically induced differentiation with (But)2cAMP and HMBA.